Acetate, lactate, propionate, and isobutyrate as electron donors for iron and sulfate reduction in Arctic marine sediments, Svalbard

Acetate, lactate, propionate, and isobutyrateas electrondonors forironand sulfate reduction inArcticmarine sediments, SvalbardNiko Finke, Verona Vandieken & Bo Barker Jørgensen

Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Bremen, Germany

Correspondence: Niko Finke, Exobiology

Branch, NASA Ames Research Center, Mail

Stop 239-4, Moffett Field, CA 94035-1000,

USA. Tel.: 11 650 6041230; fax: 11 650

6041088; e–mail: [email protected]

Present address: Niko Finke and Verona

Vandieken, Exobiology Branch, NASA Ames

Research Center, Moffett Field, CA, USA

Received 25 January 2006; revised 17 July

2006; accepted 10 August 2006.

First published online 27 October 2006.

DOI:10.1111/j.1574-6941.2006.00214.x

Editor: Alfons Stams

Keywords

volatile fatty acids; potential selenate reduction;

inhibition; acetate turnover.

Abstract

The contribution of volatile fatty acids (VFA) as e–-donors for anaerobic terminal

oxidation of organic carbon through iron and sulfate reduction was studied in

Arctic fjord sediment. Dissolved inorganic carbon, Fe21, VFA concentrations, and

sulfate reduction were monitored in slurries from the oxidized (0–2 cm) and the

reduced (5–9 cm) zone. In the 0–2 cm layer, 2/3 of the mineralization could be

attributed to sulfate reduction and 1/3 to iron reduction. In the 5–9 cm layer,

sulfate reduction was the sole mineralization process. Acetate and lactate turnover

rates were measured by radiotracer. Inhibition of sulfate reduction with selenate

resulted in the accumulation of acetate, propionate, and isobutyrate. The acetate

turnover rates determined by radiotracer and accumulation after inhibition were

similar. VFA turnover accounted for 21% and 52% of the mineralization through

sulfate reduction in the 0–2 and 5–9 cm layer, respectively. Acetate and lactate

turnover in the inhibited 0–2 cm slurry was attributed to iron reduction and

accounted for 10% and 2% of the iron reduction. Therefore, 88% and 79% of the

iron and sulfate reduction in the 0–2 cm layer, respectively, must be fueled by

alternative e–-donors. The accumulation of VFA in the selenate–inhibited 0–2 cm

slurry did not enhance iron reduction, indicating that iron reducers were not

limited by VFA availability.

Introduction

Anaerobic degradation of complex organic material in

aquatic systems is a multi-step process involving a large

diversity of physiologically specialized microorganisms (e.g.

Blackburn, 1987; Capone & Kiene, 1988). The metabolic

products of fermentative bacteria serve as electron donors

for the terminal oxidizing bacteria that use inorganic

electron acceptors for the complete oxidation of the organic

matter. In marine sediments, iron reduction and sulfate

reduction are generally the most important terminal oxida-

tion processes in the upper anoxic zone (Thamdrup, 2000).

Microorganisms that reduce iron and sulfate may use a

broad range of electron donors, yet the list of potential

substrates provides little information about the substrates

used in situ by these organisms. The substrates used by

sulfate-reducing bacteria in marine sediments have been

determined mainly by two methods: the substrate turnover

has been measured using radiolabeled substrates (Christen-

sen & Blackburn, 1982; Shaw et al., 1984; Sansone, 1986;

Shaw & McIntosh, 1990; Wellsbury & Parkes, 1995) or the

accumulation of substrates has been measured after inhibi-

tion of the sulfate reduction by molybdate (S�rensen et al.,

1981; Parkes et al., 1989; Shaw & McIntosh, 1990; Fukui

et al., 1997). These investigations have shown that volatile

fatty acids (VFA), and in particular acetate, together with

hydrogen are the major substrates for sulfate reduction.

Similar investigations for iron reduction or simultaneous

iron and sulfate reduction are lacking for marine sediments.

Furthermore, most of these studies were done in temperate

sediments and little is known about the substrates for sulfate

reducers in permanently cold sediments, which account for

4 90% of the ocean floor (Levitus & Boyer, 1994).

Molybdate is a commonly used inhibitor of sulfate

reduction. Unfortunately, it complexes VFA (Rosenheim,

1893; Finke, 1999) and thus prevents their determination by

the common HPLC technique based on 2-nitrophenyl hydra-

zine derivatization (Mueller Harvey & Parkes, 1987; Albert

& Martens, 1997). As an alternative to molybdate, selenate

can be used as a specific inhibitor of sulfate reduction

(Oremland & Capone, 1988) and allows subsequent deriva-

tization with 2-nitrophenyl hydrazine (Finke, 1999).

We investigated the relative contributions of iron reduc-

tion and sulfate reduction to the terminal oxidation of

FEMS Microbiol Ecol 59 (2007) 10–22c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

organic carbon in permanently cold Arctic sediments. More

specifically, we combined VFA turnover measurements

using radiotracer incubations with sulfate reduction inhibi-

tion studies using selenate to determine the importance of

acetate, lactate, propionate, and isobutyrate as electron

donors for iron and sulfate reduction. To our knowledge,

this is the first study of the contribution of VFA as substrates

for iron-reducing bacteria in marine sediments.

Materials and methods

Sediment

Sediment samples were taken with a HAPS corer at Station J

(79142.006N 11105.199E) in Smeerenburgfjorden on the north-

west coast of Svalbard, northern Barents Sea, in August 2004.

The in situ temperature was 2.3 1C and the water depth 212 m.

The sediment and its microbiology are described in further

detail in Vandieken et al. (2006) and Arnosti et al. (2005).

Incubations

Sediment from each of the two depth intervals, 0–2 cm and

5–9 cm, was mixed with an equal amount of anoxic seawater,

homogenized under N2, filled in two glass bottles, and sealed

with butyl stoppers. A sodium selenate solution was added

to one parallel for each depth (‘0–2 cm Se’ and ‘5–9 cm Se’)

to a final concentration of 5 mM. In our previous experi-

ments with Svalbard sediments, this concentration had

proven sufficient to completely inhibit sulfate reduction.

The slurries were incubated at 0 1C for 28 days. At 12 time

points between 1 h and 28 days after start of the incubation,

subsamples were taken for sulfate reduction rate (SRR)

measurement and pore water sampling.

Pore water analyses

Pore water for the determination of Fe21, Mn21, Ca21,

volatile fatty acids sulfide, sulfate, selenate, selenite, and

dissolved inorganic carbon (DIC) was obtained by centrifu-

ging sediment samples in glass centrifuge tubes without head-

space for 10 min at 2500 g at 4 1C. Pore water for volatile fatty

acids (VFA) analysis was obtained by centrifugation in Spinexr (Phenomenex) filter units at 2500 g at 4 1C for 10 min.

DIC was analyzed by flow injection with conductivity

detection (Hall & Aller, 1992). Fe21 was measured spectro-

photometrically (Shimadzu UV 1202) at 562 nm with Fer-

rozine (1 g L�1 in 50 mM HEPES buffer, pH 7) according to

Stookey (1970). Mn21 and Ca21 were measured by induc-

tively coupled plasma atomic emission spectrometry (Perkin

Elmer Optima 3300 RL). Sulfate was measured by non-

suppressed ion chromatography (Waters, column IC-

PakTM, 50� 4.6 mm) (Ferdelman et al., 1997). Sulfide was

determined by the methylene blue spectrophotometric

method (Shimadzu, UV 1202) at 670 nm (detection limit

1 mM) (Cline, 1969). To determine the selenate reduction

rate as a result of selenate addition, selenate and selenite

concentrations were analyzed by anion chromatography

(Dionex DX500, eluent: 9 mM NaCO3, precolumn: AG9

HC, column: AS9 HC). The detection limit was 0.2mM for

both selenate and selenite. Volatile fatty acids were measured

after derivatization with 2-nitrophenyl hydrazine by absor-

bance at 400 nm on an HPLC (Albert & Martens, 1997),

mostly in single samples but with duplicate determinations

at selected time points. VFA measured with this method

comprise acetate, propionate, lactate, and isobutyrate.

From the mean concentration change over time, produc-

tion and consumption rates for DIC, iron and selenate were

calculated using a regression line. The standard deviation was

calculated as deviation from the linear regression over time.

Sulfate reduction rates

At each time point, duplicate subsamples of the slurries were

incubated with 50 kBq 35S-sulfate. After 6 h the incubations

were stopped with 20% ZnAc and frozen. The 35S-labeled

reduced sulfur fraction was extracted using the cold chro-

mium distillation method (Kallmeyer et al., 2004) in single

samples for most time points. SRR were calculated as

described by J�rgensen (1978).

Volatile fatty acid turnover rates

After 1, 4, 8, and 14 days of incubation, subsamples of the

slurries were taken to measure VFA turnover rates. Approxi-

mately 10 mL of slurry was filled into N2 flushed syringes.

Tracer solutions were prepared in sterile filtered, anoxic pore

water at least 1 h before incubation. 14C2-acetate or 14Cu-

lactate 300 kBq was injected into triplicate syringes for each

slurry. After 10, 20, 30, 40, 50, and 60 min, c. 1.5 mL of the

sample was withdrawn into 5 mL 2% NaOH. This served to

stop the reaction and fix the 14C-DIC produced. Blank

samples were prepared by addition of the tracer to the NaOH

before addition of the sediment. 14C-TIC and 14C-acetate/

lactate were separated by the shaker method (Joye et al., 2004).

In brief, 6 M HCl was added to the sample to drive out the

DIC as CO2, which was trapped in phenylethylamine/NaOH.

The trapped 14C-DIC and the remaining 14C in the sample

were measured by scintillation counting. The turnover rate

constant was determined as the slope of the fraction of tracer

turned over per time. Multiplication with the measured

concentration yielded the turnover rate for each organic acid.

Acetate, propionate, and isobutyrate oxidation rates

coupled to sulfate reduction were calculated for the unin-

hibited slurries from the accumulation of the fatty acid after

inhibition of sulfate reduction with selenate. The accumula-

tion rates in the uninhibited slurries were subtracted from

the rates in the inhibited slurries, thus showing the

FEMS Microbiol Ecol 59 (2007) 10–22 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

11VFA as substrates for iron and sulfate reduction

oxidization rate by sulfate reduction in the uninhibited

slurries. The standard deviation was calculated as the devia-

tion from the linear regression over time.

Results

Pore water chemistry

Dissolved inorganic carbon, Ca21, Fe21, Mn21, sulfate, and

sulfide pore water concentrations were measured in anoxic

sediment slurries from 0–2 and 5–9 cm depths (uninhibited

slurries) at 12 sampling points over 28 days of incubation.

These parameters were also measured in parallel incubations

where 5 mM selenate was added to inhibit sulfate reduction

(inhibited slurries). In these incubations, pore water concen-

trations of selenate and selenite were also determined. Exam-

ple plots for DIC, Fe21, and Mn21 are shown in Fig. 1 and for

selenate and selenite in Fig. 2. DIC concentrations increased

during the incubation in all slurries (Fig. 1a). Pore water Ca21

concentrations were constant in all samples, which indicated

that carbonate precipitation did not take place during the

experiments (data not shown). Fe21 accumulated in the

pore water throughout the incubation (Fig. 1b) with rates

of 7 nmol cm�3 day�1 for the 0–2 cm slurry and

0.5 nmol cm�3 day�1 for the 5–9 cm slurry. There were no

significant differences between the inhibited and uninhibited

slurries. Mn21 concentrations stayed constant over the course

of the incubation in all slurries (Fig. 1b). Sulfate concentra-

tions did not change over time in all slurries, sulfide concen-

trations stayed below detection limit throughout the

incubation (data not shown). The added selenate in both

inhibited slurries decreased in concentration over the course

of the incubation (Fig. 2). Selenite, a potential product of

microbial selenate reduction (Oremland & Stolz, 2000), was

only detectable after 20 days, reaching 4.3mM at the end in the

5–9 cm inhibited slurry (Fig. 2). No selenite was detected

throughout the experiment in the 0–2 cm inhibited slurry.

Sulfate reduction rates

Sulfate reduction rates in the 0–2 cm slurry varied between

48 and 70 nmol cm�3 day�1 with rather constant rates after

an initial increase (Fig. 3). In the 5–9 cm slurry the sulfate

reduction rates were between 18 and 31 nmol cm�3 day�1

with increased rates of 62–70 nmol cm�3 day�1 on days 1–4

(Fig. 3). Addition of �5 mM selenate inhibited sulfate

reduction rates to below the detection limit of

2 nmol cm�3 day�1 in both depth intervals (data not shown).

At selected time points the sulfate reduction rates were

determined in duplicate samples. The second determination

resulted in rates 5–10% different from the original measure-

ment (data not shown).

Volatile fatty acids

Volatile fatty acids concentrations are shown as mM C in Fig.

4. Acetate occurred in the highest concentrations in all

Fig. 1. Time course of DIC, Fe21 and Mn21 concentrations in the pore

water of slurry from the 0–2 cm depth interval without selenate inhibi-

tion. Production rates were calculated from the time course as indicated

by the regression line.

Fig. 2. Time course of selenate and selenite concentrations in the pore

water of selenate–amended slurry from the 5–9 cm depth interval.

Selenate reduction rates were calculated from the time course as

indicated by the regression line.

FEMS Microbiol Ecol 59 (2007) 10–22c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

12 N. Finke et al.

samples, followed by propionate, lactate, and isobutyrate

(data not shown). Lactate concentrations remained around

1–2mM throughout the incubation. In the uninhibited

0–2 cm slurry the acetate concentrations increased from 45

to 78 mM after 2 days and showed a second transient increase

with maximum concentrations after 12 days (Fig. 4). The

concentrations decreased to 3–8mM towards the end of the

incubation. In the 5–9 cm uninhibited slurry the acetate

concentrations increased from 45 to 93 mM after 3 days

followed by a decrease to 3–8mM. Propionate increased

from 0.8 to 3 mM after 2 and 3 days in the 0–2 and 5–9 cm

slurry, respectively, and decreased again to values around

1 mM. Isobutyrate concentrations remained around 0.5 mM

throughout the incubation in both uninhibited slurries

(data not shown).

In the 0–2 cm inhibited slurry, the VFA concentrations

increased throughout the entire incubation, reaching 680,

60, and 22 mM for acetate, propionate, and isobutyrate,

respectively, after 28 days. In the 5–9 cm inhibited slurry,

VFA concentrations increased for the first 12 days, reaching

335, 18, and 7.5 mM for acetate, propionate, and isobutyrate,

respectively, and decreased towards the end of the incuba-

tion. Duplicate measurements at selected time points varied

by about 5% to a maximum of 10% (data not shown).

The oxidation of acetate, propionate, and isobutyrate by

sulfate reducers was determined from the accumulation in

the inhibited slurries. Acetate and propionate accumulated

at higher rates in the inhibited vs. the uninhibited slurries at

the beginning of the incubation. The difference in the

accumulation of acetate and propionate in the inhibited vs.

the uninhibited slurry and the isobutyrate accumulation

rate in the inhibited slurries was attributed to sulfate

reduction (Table 1). VFA accumulation rates were highest

for acetate followed by propionate and isobutyrate.

In the 14C-tracer incubations the rate constants for the

turnover of the acetate and lactate pools were determined

(Table 2). The constants for lactate were generally 3–15-fold

higher than for acetate, but due to higher concentrations of

acetate compared to lactate the turnover rates of acetate

were 1.5–9-fold higher in the uninhibited slurries and

3–120-fold in the inhibited slurries than for lactate (Fig. 5).

The acetate turnover rates were higher in the uninhibited

than in the inhibited slurries during the first 1- and 4-day

incubations but were similar or highest in the inhibited

slurries during the following 8- and 14-day incubations. The

lactate turnover rates were 2–10-fold higher in the unin-

hibited compared to the inhibited slurries throughout the

experiment.

Contribution of different electron acceptors todissolved inorganic carbon production

The anaerobic carbon mineralization during the incubations

was determined by measurement of DIC production. The

DIC production rates in the inhibited slurries were

Fig. 3. Sulfate reduction rates measured in the uninhibited slurries from

0–2 cm and 5–9 cm. In the selenate amended slurries the rates were

below the detection limit of 2 nmol cm�3 d�1 (data not shown).

Fig. 4. Volatile fatty acids (VFA) concentrations measured in the pore

water of the four slurries. Concentrations are shown in mM C. (a) shows

acetate concentrations and (b) the sum of lactate, propionate, isobuty-

rate.

FEMS Microbiol Ecol 59 (2007) 10–22 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

13VFA as substrates for iron and sulfate reduction

approximately half those in the uninhibited slurries (Fig. 6;

Table 3). Their contribution to the oxidation of organic

carbon and, thus, to DIC production were calculated from

the mean sulfate reduction rate and selenate reduction rate

according to the reactions given in Table 4. Sulfate reduction

accounted for 76% and 96% of the DIC production in the

0–2 and 5–9 cm slurries, respectively.

Sulfate reduction was inhibited by selenate addition and

part of the DIC production was attributed to microbial

carbon oxidation coupled to selenate reduction. In sedi-

ments, selenate is usually reduced to selenite or elemental

selenium (Se1) (Majers et al., 1988; Steinberg & Oremland,

1990; Oremland et al., 1994a; Stolz & Oremland, 1999;

Herbel et al., 2000; Lucas & Hollibaugh, 2001; Knight et al.,

2002). As selenite accumulated only towards the end of the

incubation at very low rates, reduction to Se1 was assumed

for the calculation of the contribution of selenate reduction

to DIC production (Table 3). In the 0–2 cm inhibited slurry,

selenate reduction accounted for 50% of the DIC produc-

tion. In the 5–9 cm inhibited slurry, the selenate reduction

exceeded the DIC production (Table 3), but the difference

was not larger than the uncertainty of the rates.

The DIC production not accounted for by sulfate or

selenate reduction was attributed to microbial iron reduc-

tion according to the stoichiometry given in Table 4 (Can-

field et al., 1993). The uncertainty of the iron reduction was

calculated as the sum of the uncertainties of the DIC

production and sulfate or selenate reduction. In the 0–2 cm

uninhibited slurry the calculated microbial iron reduction

rate was 60 nmol C cm�3 day�1 and iron reduction ac-

counted for 34% of the DIC production. In the 0–2 cm

inhibited slurry the rate of DIC production attributed to

iron reduction was similar to the uninhibited slurry,

41 nmol C cm�3 day�1 (Table 3). The uncertainty was larger

than the difference between the rates in the two slurries.

Thus, the inhibition of sulfate reduction did not change the

Table 1. Acetate, lactate, propionate, and isobutyrate as substrates for sulfate reduction. Turnover rates as measured at the beginning of the

incubation

AcetateLactate Propionate Isobutyrate

SumwTracer� Inhibition Tracer� Inhibition Inhibition

VFA turnover

(nmol cm�3 day�1)

0–2 cm 6.1�2.7 6.9� 1.1 3.4� 0.6 1.1� 0.2 0.42� 0.02

5–9 cm 15.1� 1.9 18.9� 1.9 1.1� 0.3 1.7� 0.1 0.19� 0.02

Contribution to

sulfate reduction (%)z

0–2 cm 10�4 12� 2 5.8� 0.9 3.3� 0.5 1.8� 0.1 20.9�5.5

5–9 cm 40�5 50� 5 2.9� 0.8 7.9� 0.3 1.3� 0.1 52.2�6.2

�Turnover attributed to sulfate reduction calculated as difference in rates measured in inhibited and uninhibited slurries.wAcetate contribution taken from tracer incubation.zContribution of turnover of the acids to sulfate reduction based on a stoichiometry of organic acid : sulfate of 1 : 1 for acetate, 1 : 1.25 for lactate,

1 : 1.75 for propionate, and 1 : 2.5 for isobutyrate assuming complete oxidation of the acid to DIC. Sulfate reduction rates were taken from Table 3.

Rates were measured with radiotracer for acetate and lactate and from accumulation after selenate inhibition for acetate, propionate and isobutyrate.

Contribution to sulfate reduction was calculated based on the stoichiometry given above. Mean and standard deviation of triplicate determinations for

the tracer incubations. Standard deviation of the inhibition experiment based on the deviation of the concentration time course from a straight

regression line.

Table 2. Turnover rate constants (h�1) for acetate and lactate as measured in the radiotracer incubations

Day 1 Day 4 Day 8 Day 14

Acetate

0–2 cm 0.009� 0.002 0.015� 0.002 0.015� 0.005 0.015� 0.003

0–2 cm inhibited 0.0013�0.0006 0.0013� 0.0005 0.0010�0.0001 0.0013� 0.0012

5–9 cm 0.012� 0.001 0.0093� 0.0019 0.030� 0.003 0.063� 0.016

5–9 cm inhibited 0.00085�0.00007 0.0017� 0.001 0.00077�0.00026 0.0013� 0.0005

Lactate

0–2 cm 0.25� 0.04 0.17� 0.020 0.21� 0.040 0.32� 0.15

0–2 cm inhibited 0.028� 0.004 0.051� 0.009 0.031� 0.012 0.031� 0.001

5–9 cm 0.072� 0.005 0.12� 0.03 0.073� 0.015 0.22� 0.15

5–9 cm inhibited 0.020� 0.008 0.038� 0.017 0.012� 0.004 0.038� 0.016

Mean and standard deviations of three parallel determinations

FEMS Microbiol Ecol 59 (2007) 10–22c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

14 N. Finke et al.

iron reduction within the accuracy of our method. This was

supported by similar rates of Fe21 accumulation in the pore

water in both incubations. In the uninhibited 5–9 cm slurry

the calculated iron reduction rates of 3 nmol cm�3 day�1

were less than the sum of the uncertainties of the sulfate

reduction and DIC production. In agreement with the fact

that no HCl-extractable Fe(III) was available in this sedi-

ment horizon (Vandieken et al., 2006), we concluded that

sulfate reduction was the sole electron accepting process.

Discussion

Mineralization of organic matter

Anaerobic bacteria performing the terminal oxidation of

organic carbon to CO2 in marine sediments rely on hydro-

lytic and fermentative bacteria to degrade complex polymers

into small organic molecules. The most important electron

acceptors below the oxic zone in coastal sediments are iron

oxides and sulfate (Thamdrup, 2000). In a range of marine

environments from temperate to permanently cold sedi-

ments, microbial iron reduction accounted for 0–75% of

anaerobic carbon oxidation (S�rensen, 1982; Canfield &

Marais, 1993; Thamdrup & Canfield, 1996; Kostka et al.,

1999, 2002; Glud et al., 2000; Kristensen et al., 2000;

Thamdrup, 2000; Jensen et al., 2003). Comparison of rates

measured at different in situ temperatures indicates that

temperature does not control the relative importance of iron

and sulfate reduction (Thamdrup, 2000). The concentration

of poorly crystalline iron oxides seems to be important for

the competition between iron and sulfate reducers and, thus,

determines the relative importance of these two processes

(Thamdrup, 2000; Jensen et al., 2003; Vandieken, 2005). As a

result, the vertical separation between these oxidation

processes is usually not complete and microbial iron reduc-

tion often co-occurs with sulfate reduction (Vandieken

et al., 2006; S�rensen, 1982; Canfield et al., 1993; Thamdrup

& Canfield, 1996; Kristensen et al., 2000; Kostka et al., 2002).

HCl-extractions of Smeerenburgfjorden sediment showed

reactive Fe(III) in the top 2 cm (Vandieken et al., 2006),

resulting in concurrent iron and sulfate reduction in the

0–2 cm layer (Table 3). In the 5–9 cm layer sulfate reduction

was the sole detectable terminal oxidation step. The rates of

microbial iron reduction and sulfate reduction in this study

(Table 3) were similar to rates determined previously for this

sediment (Vandieken et al., 2006).

Selenate reduction

Selenate reduction potential was previously detected in

sediments ranging from polluted to pristine sites by

Fig. 5. 14C-acetate and 14C-lactate turnover rates measured in the

0–2 cm, 0–2 cm Se, 5–9 cm, and 5–9 cm Se slurries. The rates are given

in mol C produced during substrate oxidation. The open circles in the

acetate turnover graph represent the rates calculated from the differ-

ence in the acetate accumulation in the inhibited compared to the

uninhibited slurries. Mean and standard deviations from triplicate in-

cubations.

Fig. 6. Dissolved inorganic carbon (DIC) production in the 0–2 cm,

0–2 cm Se, 5–9 cm, and 5–9 cm Se slurries. Actual dissolved inorganic

carbon production as calculated from dissolved inorganic carbon accu-

mulation and theoretical production as calculated from sulfate reduction

and selenate reduction according to the reactions shown in Table 4.

Standard deviations are based on the deviation of the concentration time

course from a linear regression.

FEMS Microbiol Ecol 59 (2007) 10–22 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

15VFA as substrates for iron and sulfate reduction

following the reduction of added selenate (Steinberg &

Oremland, 1990; Lucas & Hollibaugh, 2001). The solid

phase selenium concentrations in Smeerenburgfjorden sedi-

ments were below our detection limit of 16 ppb (M.

Isenbeck-Schroter, unpublished data). Selenate can be mi-

crobially reduced to selenite, Se1, or selenide (Stolz &

Oremland, 1999; Lloyd et al., 2001). In bacterial cultures

and in aquatic sediments selenate is usually reduced to

selenite or Se1 (Majers et al., 1988; Steinberg & Oremland,

1990; Oremland et al., 1994a; Stolz & Oremland, 1999;

Herbel et al., 2000; Lucas & Hollibaugh, 2001; Knight et al.,

2002). Selenite accumulated only towards the end of our

incubation in the 5–9 cm slurry (Fig. 2). Thus, we assume

that organic matter was mainly coupled to the reduction of

selenate to Se1 in both inhibited slurries according to the

reaction given in Table 4. The potential selenate reduction

rates of 27 and 37 nmol cm�3 day�1 in the 0–2 and 5–9 cm

layer, respectively, lie within the reported range of potential

selenate reduction rates in pristine and contaminated sedi-

ments of 1.7 to 530 nmol cm�3 day�1 (Steinberg & Orem-

land, 1990; Lucas & Hollibaugh, 2001).

By comparing the DIC production in the uninhibited and

inhibited slurries, the major effects of selenate addition

seemed to be inhibition of sulfate reduction and initiation

of selenate reduction. The time course of Fe21 and Mn21

concentrations were the same in the inhibited and unin-

hibited slurries. Therefore, a general toxic effect of the added

high selenate concentrations to the microbial community

was not evident.

Volatile fatty acids

Acetate turnover measurements

The importance of acetate as substrate for sulfate-reducing

bacteria in marine sediments has been investigated pre-

viously using radiotracer and molybdate inhibition experi-

ments (Ansbaek & Blackburn, 1980; Sansone & Martens,

1981; Christensen & Blackburn, 1982; Winfrey & Ward,

1983; Christensen, 1984; Shaw et al., 1984; Shaw & McIn-

tosh, 1990). To evaluate the contribution of acetate as an

electron donor for sulfate reduction, radiotracer incubations

were performed in sediments with sulfate being the sole

terminal oxidation process. These measurements often lead

to a discrepancy between sulfate reduction and acetate

turnover rates with measured acetate turnover rates exceed-

ing sulfate reduction rates or total mineralization based on

DIC or NH41 production (Ansbaek & Blackburn, 1980;

Sansone & Martens, 1981; Christensen & Blackburn, 1982;

Shaw et al., 1984; Wu & Scranton, 1994). In contrast,

molybdate inhibition experiments gave acetate turnover

rates (calculated from the accumulation of acetate) that

were lower than sulfate reduction rates or total mineraliza-

tion (S�rensen et al., 1981; Winfrey & Ward, 1983;

Table 3. Dissolved inorganic carbon production in the 0–2 cm, 0–2 cm inhibited (with selenate addition), 5–9 cm, and 5–9 cm inhibited (with selenate

addition) slurries

Total DIC production Sulfate reduction Selenate reduction Calculated iron reduction Iron accumulation

0–2 cm 175� 8 115�16 – 60� 24 34%� 1.7�0.2

0–2 cm inhibited 82� 13 o 2 41�17 41� 30 50%� 1.9�0.1

5–9 cm 79� 8 76�13 – 3� 21 0% w 0.16�0.03

5–9 cm inhibited 41� 4 o 2 56�12 0 0% 0.11�0.09

�Relative contribution of iron reduction to DIC production.wContribution set to 0% as the uncertainty is larger than the calculated rate.

DIC, dissolved inorganic carbon.

Contribution of the different electron accepting processes to the production of dissolved inorganic matter was calculated on a C-molar basis

(nmol C cm�3 day�1) according to the reactions shown in Table 4. The values represent a mean of the 28 days of incubation. Standard deviations are

based on the deviation of the concentration time course from a linear regression line

Table 4. Terminal oxidation reactions of organic carbon and acetate using different electron acceptors

Electron donor Electron acceptor Reaction

Stoichiometry

Electron donor:acceptor

Organic carbon Sulfate 2CH2O1SO42– ! 2HCO3

–1HS–1H1 2 : 1

Organic carbon Iron(III) CH2O14Fe(OH)3 ! HCO3–14Fe2113H2O17OH– 1 : 4

Organic carbon Selenate 3CH2O12SeO42– ! 3HCO3

–12Se11H112OH– 3 : 2

Acetate Sulfate CH3COO–1SO42– ! 2HCO3

–1HS– 1 : 1

Acetate Iron CH3COO–18Fe(OH)3 ! 2HCO3–18Fe2115H2O115OH– 1 : 8

Organic carbon is represented as CH2O with an oxidation state of 0 for carbon

FEMS Microbiol Ecol 59 (2007) 10–22c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

16 N. Finke et al.

Christensen, 1984; Shaw & McIntosh, 1990). The higher

acetate turnover rates measured in the radiotracer studies

probably were a result of an overestimation of the bioavail-

able acetate pool. It was suggest that some of the pore water

acetate was complexed and less bioavailable, whereas the

tracer was added in a free form and thus was readily taken

up by the microorganisms (e.g. Shaw & McIntosh, 1990;

Kristensen et al., 1994). Christensen & Blackburn (1982)

reported decreasing acetate turnover rate constants for

incubation times of more than 10 min, probably due to

partitioning of the tracer in the different acetate pools. The

rates determined with shorter incubation times, however,

exceeded total mineralization based on NH41-production by

five-fold.

In the present study, the tracer solution was prepared in

sterile-filtered, anoxic pore water at least 1 h before injec-

tion. Thus, we assume that the tracer should be complexed

just as the acetate in the pore water, and the turnover rate

constants measured should reflect the turnover of the total

acetate pool. From the oxidation rates determined with this

method, acetate oxidation coupled to sulfate reduction can

be calculated from the difference of the tracer turnover in

the inhibited and uninhibited slurry (Fig. 5). The acetate

turnover rates measured with the radiotracer were not

significantly different from the accumulation rates in the

inhibited slurries (Table 1) and accounted for 10% and 40%

of the sulfate reduction in the 0–2 cm and 5–9 cm layer,

respectively, based on the reactions shown in Table 4. This

supports the suggestion that complexation of acetate was

responsible for the overestimation of turnover rates in

previous studies.

Volatile fatty acids as substrate for terminaloxidation

The inhibition experiment showed that acetate was the most

important of the VFA as a substrate for sulfate reducers, as

seen by the highest accumulation rate (Table 1). The only

other VFA accumulating after inhibition were propionate

and isobutyrate but at lower rates than acetate.

The turnover rates calculated from VFA accumulation

are only appropriate if the selenate reducers did not oxidize

these acids at similar rates. Isolated selenate-reducing

organisms used acetate and lactate as electron donors in

pure culture studies (Oremland et al., 1994b, 1989; Stolz &

Oremland, 1999; Oremland & Stolz, 2000). Addition of

acetate to sediment slurries enhanced selenate reduction

(Oremland et al., 1989). In our experiment the lactate and

acetate turnover was initially strongly reduced in the

inhibited slurries (Fig. 5), indicating that selenate-reducing

bacteria did not use acetate and lactate as their major

substrates at the beginning of the incubation. The accumu-

lation rates of propionate and isobutyrate were 7–10 and

17–90 times lower than for acetate, a similar ratio to that

found in previous investigations (Table 5). However, due to

the high variability in reported turnover rates it is difficult

to evaluate whether the selenate reducers oxidized these

acids.

Many sulfate reducers are known to utilize lactate (Rabus

et al., 2000). Surprisingly, lactate did not accumulate in the

inhibited slurries, even though tracer incubations showed

that the oxidation was strongly inhibited (Fig. 5). The fact

that there is an almost complete inhibition of lactate turn-

over in the first tracer incubations in the inhibited slurries

suggests that lactate served as substrate for sulfate reducers.

Apparently, its production is strongly reduced in the in-

hibited slurry. Thus, we attribute the lactate turnover

measured in the tracer incubation to sulfate reduction.

Together, acetate, lactate, propionate, and isobutyrate ac-

counted for 21% and 52 % of the sulfate reduction in the

0–2 cm and 5–9 cm slurries, respectively (Table 1).

Christensen (1984) reported a contribution of 65% for

acetate and 5% and 8% for propionate and isobutyrate to

sulfate reduction in Danish coastal sediments. (Table 5).

Parkes & Jorck-Ramberg (1984) reported that VFA ac-

counted for 4 75% of the substrates for sulfate reduction

in temperate marine and estuarine sediments. In Smeeren-

burgfjorden sediment, acetate was the most important

electron donor of the investigated VFA, followed by lactate

and propionate, and finally isobutyrate (Table 1). In the

5–9 cm layer, which was dominated by sulfate reduction, the

42% contribution of acetate to sulfate reduction was similar

to previous investigations using molybdate inhibition (Table

5). In the 0–2 cm zone, where sulfate reduction and iron

reduction occurred simultaneously, acetate was much less

important as an electron donor. Based on the stoichiometry

given in Table 4, only 10% of the sulfate reduction could be

attributed to acetate oxidation (Table 1).

Iron-reducing bacteria are very diverse and may use a

wide range of electron donors (Coates et al., 1996, 1998;

Anderson et al., 1998; Kashefi et al., 2003). To date there are

no investigations of substrates for iron reducers in marine

sediments. In a previous study of substrates for iron

reducers in a freshwater sediment, addition of 14C-labeled

glucose showed that acetate is the most important inter-

mediate in glucose degradation (Lovley & Phillips, 1989).

Roden & Wetzel (2003) identified acetate as important

substrate for iron reducers in freshwater sediments.

The maximum contribution of acetate and lactate turn-

over to iron reduction can be calculated from the acid

turnover not attributed to sulfate reduction. Acetate accu-

mulation in the inhibited vs. the uninhibited slurry was the

same as the turnover rates determined with radiotracer

incubations (Table 1). This indicates that the selenate

reducers were not important for acetate oxidation at the

beginning of the incubation. The acetate turnover

FEMS Microbiol Ecol 59 (2007) 10–22 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

17VFA as substrates for iron and sulfate reduction

in the inhibited slurry was attributed to iron reduction.

Lactate did not accumulate in the inhibited slurries, making

it more difficult to evaluate the effect of the selenate addi-

tion on lactate turnover. However, attributing all lactate

turnover in these slurries to iron reduction gave a maxi-

mal contribution of lactate to iron reduction. Altogether,

only 10% and 2% of the iron reduction in the 0–2 cm

layer could be attributed to acetate and lactate oxida-

tion, respectively. If part of the acetate and lactate was

oxidized by selenate reducers, the contribution would be

even lower.

The increased acetate concentrations in the selenate

inhibited slurry did not enhance the iron reduction (Table

3). If acetate was the dominant electron donor for the iron

reducers and the iron reduction was electron donor limited,

increasing the acetate concentration should stimulate iron

reduction. The absence of a stimulation of the iron reduc-

tion after increase in VFA concentrations shows that the iron

reducers used other electron donors or were not electron

donor limited.

In conclusion, 88% and 79% of the iron and sulfate

reduction, respectively, in the 0–2 cm layer must be driven

Table 5. Compilation of acetate (Ac), lactate (La), propionate (Pro), and isobutyrate (iB) concentration and turnover measurements in sulfate-reducing

marine sediments

Location

VFA

concentration

(mM)

Acetate turnover

rate constants (h�1)

VFA oxidation rate

(nmol cm�3 day�1)

Sulfate reduction

(nmol cm�3 day�1)

Temperature

( 1C) Technique Reference

Mangrove, Thailand Ac 0.5–31 0.96–5.8 33–1200 5–130 28/33 3H tracer Kristensen et al. (1994)

Cape Lookout Bight,

USA

Ac 54–700�

La 4–32�

Pro 1–24�

iB 0.2–6�

0.5–6.2 423–13000

65–1200

17–170

480–1200 6–28 14C tracer Sansone & Martens

(1981, 1982), Sansone

(1986), SRR: Martens &

Klump (1980)

Loch Eil, Loch Etive, and

Tay Estuary, Scotland

Ac 14.3–35.5 0.006–0.22w 2.3–272z 1.2–1560 25 MoO42–

inhibition

Parkes et al. (1989)

La 3.5–24 0–155z

Pro 1.3–2.4 0–34z

iB 0–0.02 2.9–8.8z

Tamar Estuary, UK Ac 2–20 0.27–0.69 20–250 15–40 25 14C tracer Wellsbury & Parkes

(1995)

Coastal lagoon Ac 5 4.3w 480–650‰ 980–1300‰ 20 MoO42–

inhibition

S�rensen et al. (1981)

Pro 1 72–170‰

iB 1 29–58‰

Ise Bay, Japan Ac 30–140 0.069–0.17w 200–363 50–210 �24z MoO42–

inhibition

Fukui et al. (1997)

Proo 1–43 13–16 0–9.7

iBo 1–8.1

Danish coastal waters Ac 2–70 1–13 500–8500 (16% of VFA

oxidation rate)

3.5/12 14C tracer Christensen & Blackburn

(1982)

Skan Bay, Alaska Ac 3.1–10.8 0.77–1.7w 57.6–400z 12–72z 4 14C tracer (Shaw et al. (1984)

Skan Bay Alaska Ac 1.1–13.8 1 26–36 50 4 14C tracer,

MoO42–

inhibition

Shaw & McIntosh

(1990)

Limfjorden, Denmark Ac 0.1–6 1–4.8 24–288 (33% of VFA

oxidation rate)

2/7 14C tracer Ansbaek & Blackburn

(1980)Pro 0.04–0.19

Coastal lagoon,

Denmark

Ac 15–40 0.095–0.15w 30–77 50–120 0 MoO42–

inhibition

Christensen (1984)

Pro 3–8

iB 0.5–2

Smeerenburgfjorden,

Svalbard (0–2 cm /

5–9 cm)

Ac 15–40 0.009–0.012 8.2/16 58/38 0 14C tracer,

selenate

inhibition

This study

La 0.8–2.7

P 0.5–1.1

iB 0.11–0.24

1.6/3.7

1.1/1.7

0.42/0.19

VFA, volatile fatty acids.�Concentrations given as nmol cm�3.wCalculated from turnover rate and concentration given.zTurnover given as mM day�1.‰Turnover given as nmol g�1 day�1.zWater temperature.

FEMS Microbiol Ecol 59 (2007) 10–22c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

18 N. Finke et al.

by electron donors other than the investigated VFA. Poten-

tial substrates for iron and sulfate reducers range from small

to larger molecules, such as hydrogen, short alcohols and

VFA, longer alcohols, and fatty acids (Rabus et al., 2000;

Lovley et al., 2004), sugars (Coates et al., 1998; Sass et al.,

2002; Kashefi et al., 2003), amino acids (Stams et al., 1985;

Kashefi et al., 2003), and aromatic (Widdel, 1980; Anderson

et al., 1998) and aliphatic hydrocarbons (Aeckersberg et al.,

1991). Parkes et al. (1989) found that amino acids accounted

for up to 10% of the sulfate reduction in marine sediments.

Investigations on other potential substrates for iron and

sulfate reducers are not available. To our knowledge our

study is the first investigation of substrates for iron reduc-

tion in marine sediments and the first on substrates for

sulfate reducers with co-occurring iron reduction. It re-

mains unclear, whether the low contribution of VFA to

terminal oxidation is related to the co-occurrence of the two

processes.

Effect of temperature

Investigations on organic matter degradation in temperate

sediments reported decreasing overall rates with decreasing

temperatures in temperate sediments (J�rgensen &

S�rensen, 1985; Crill & Martens, 1987; Westrich & Berner,

1988). In contrast, permanently cold sites did not show

generally lower rates than temperate sediments at higher

temperatures (Vandieken et al., 2006; Sagemann et al., 1998;

Thamdrup & Fleischer, 1998; Knoblauch & Jorgensen,

1999). Several investigations on bacteria in seawater have

shown a decreased substrate affinity at low temperatures

and, thus, a requirement for higher concentrations of

organic substrates (Wiebe et al., 1992, 1993; Nedwell,

1999). Weston & Joye (2005) examined the effect of chan-

ging temperatures on the coupling of fermentation and

terminal oxidation. At temperatures below 25 1C, potential

fermentation rates exceeded potential terminal oxidation

rates, resulting in increased VFA concentrations. A seasonal

investigation on VFA concentration in Cape Lookout Bight

sediments showed low VFA concentrations in winter (San-

sone & Martens, 1982). Investigations of VFA concentra-

tions from Svalbard and the German Wadden Sea showed a

close coupling of fermentation and sulfate reduction, result-

ing in constantly low VFA concentrations between 01 and

25–30 1C (Finke, 2002). Thus, in marine sediments not

amended with complex organic matter, a decoupling of the

fermentation and terminal oxidation yielding higher in situ

concentrations at low temperatures does not seem to occur.

The acetate turnover rates measured in this study at 0 1C

(Table 1) were similar to rates reported in previous studies

using the molybdate inhibition technique, being 4–7 times

higher than the lowest reported rates from Scottish costal

sediment at 25 1C (Parkes et al., 1989) and half the rates

found in Arctic and cold temperate sediments (Christensen,

1984; Shaw & McIntosh, 1990). The highest acetate turnover

rates were measured with the 14C-tracer technique in

organic rich Cape Lookout Bight sediments at 28 1C (San-

sone & Martens, 1982) and in Danish coastal waters at 8 1C

(Christensen & Blackburn, 1982). However, these rates were

probably too high due to overestimation of the acetate pool.

Measured turnover rates of VFA might not reflect the

actual biogeochemistry of the sediments due to uncertainty

of the VFA pool size. The turnover rate constant is a more

robust parameter to compare different investigations. Wu

et al. (1997) found a close coupling of the turnover rate

constant to in situ temperature in Long Island Sound

sediments. In contrast, similar maximum rate constants in

late winter and late summer were found in Cape Lookout

Bight sediments (Sansone & Martens, 1982). The highest

turnover rate constants were found in Danish coastal sedi-

ments at temperatures around 8 1C (Christensen & Black-

burn, 1982), followed by Cape Lookout Bight at 25 1C

(Sansone, 1986). The rate constants found in this study

(Table 2) were similar to the lowest reported rates found at

Loch Etive at 25 1C (Parkes et al., 1989) but were 10 times

lower than found in Danish lagoon sediments at the same

temperature (Christensen, 1984) and 100 times lower than

in the permanently cold Skan Bay sediments (Shaw et al.,

1984; Shaw & McIntosh, 1990). Thus, parameters other than

in situ temperature seem to be more important in determin-

ing the VFA turnover rate and turnover rate constant.

Acknowledgements

We thank Maren Nickel and Jacqueline Schmidt for help

with the sampling and Kristina Burkert for help with 14C

analysis. Thanks to Martina Alisch for measuring samples

with the Dionex and Silvana Hessler at the University of

Bremen for measuring samples with the ICP-AES. We thank

Margot Isenbeck-Schroter from the University Heidelberg

for determining the solid phase selenate. We thank the

skipper, Stig Henningsen, and the first mate, John Morten-

sen, for help on board the MS FARM. We thank the

University Centre on Svalbard (UNIS) for providing labora-

tory space. This study was funded by the Max Planck

Society.

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